Ref ID : 153
Eduardo Orias, Hashimoto Naoko, Miu-Fun Chau, and Higashinakagawa Toru; PCR Amplification of Tetrahymena rDNA Segments Starting with Individual Cells. J.Protozool. 38(4):306-311, 1991
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To facilitate studies of rDNA molecular genetics in Tetrahymena thermophila, we attempted the detection of polymorphisms in the nontranscribed spacers (NTSs) using polymerase chain reaction (PCR), starting with minute amounts of DNA. The targeted polymorphic regions are 85% adenine-thymine (AT). We found conditions of efficient and specific in vitro amplification of targeted segments in the replication domain of the 5'NTS and in the subtelomeric segment of the 3'NTS. The identity of the amplified segments was confirmed by restriction enzyme digestion and DNA sequence analysis. Digestion of the template DNA at restriction sites upstream and downstream of the targeted region increased the efficiency of amplification, presumably because the targeted segments are in a palindromic molecule. Starting from total cell DNA corresponding to as little as 0.03 picogram (equivalent to the DNA content of 0.003 cells or about 30 rDNA molecules), we observed the amplified band after agarose gel electrophoresis and ethidium bromide staining. The yield indicated more than 10-billion-fold amplification. Amplification of the subtelomeric fragment yielded homogeneous product of minimum possible length even though the telomeric-specific primer can bind, at least initially, at a multiplicity of GGGGTT repeats. Amplified 5'NTS product also was detected in an ethidium-bromide-stained gel when PCR was started with a single cell.